Riemerella anatipestifer double-gene deletion attenuated vaccine candidate strain, construction method and application

Abstract

The invention discloses a riemerella anatipestifer double-gene deletion attenuated vaccine candidate strain, a construction method and application, the riemerella anatipestifer double-gene deletion attenuated vaccine candidate strain is a riemerella anatipestifer CH-1 strain B739RS01935-B739RS07625 double-gene deletion attenuated vaccine candidate strain, the preservation number is CCTCC NO: M20251080, the preservation date is May 22, 2025, and the preservation number is CCTCC NO: M20251080. The strain is preserved in China Center for Type Culture Collection, and the address of the preservation unit is Wuhan University, Wuhan, China. After ducklings are immunized by the low virulent strain, the low virulent strain has no obvious influence on weight proliferation of the ducklings and has good safety. And after the ducklings are immunized, the challenge protection rate on RACH-1 wild virulent strains reaches 83.3%, and the immune protection effect is good.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to a candidate strain of a Riemerella anatipestifer double-gene deleted attenuated vaccine, a construction method and an application thereof. Background Art

[0002] Riemerella anatipestifer ( <h2 style=";text-align:left;direction:ltr">Riemerellaanatipestifer Rhizoctonia solani (RA) is the primary pathogen causing duck serositis, infecting ducks, geese, and other poultry, and is currently showing signs of spreading to chickens. The disease is widespread and has become a serious bacterial infectious disease posing a serious threat to my country's duck farming industry. Currently, the disease is primarily controlled through inactivated vaccines and medication. The preparation of inactivated vaccines is complex; medications can easily lead to bacterial resistance and environmental pollution. Furthermore, with the current trend toward reducing and restricting antibiotic use, the use of antimicrobial drugs is strictly restricted. Therefore, the development of a genetically engineered, attenuated vaccine that can be administered once and offers a strong immune response is urgently needed.

[0003] In view of this, this patent application is filed. Summary of the Invention

[0004] To solve the above problems, the present invention provides a candidate strain of a double-gene deleted attenuated vaccine of Riemerella anatipestifer, a construction method and application thereof, and also provides an attenuated vaccine for Riemerella anatipestifer disease.

[0005] The following technical solutions are specifically used: The first object of the present invention is to provide a candidate strain of attenuated vaccine of Riemerella anatipestifer with double gene deletion, which is Riemerella anatipestifer CH-1 strain <h2 style=";text-align:left;direction:ltr"> B739_RS01935-B739_RS07625 A candidate strain of the double-gene-deleted attenuated vaccine, Riemerella anatipestifer CH-1Δ <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Δ <h2 style=";text-align:left;direction:ltr"> B739_RS07625 (Classification name: Riemerella anatipestifer RACH-1ΔB739_RS01935ΔB739_RS07625), the deposit number is CCTCC NO: M20251080, the deposit date is May 15, 2025, and it is deposited in the China Center for Type Culture Collection, and the depository address is Wuhan University, Wuhan, China.

[0006] The second object of the present invention is to provide a method for constructing the above-mentioned candidate strains of attenuated vaccine of Riemerella anatipestifer with double gene deletion, including the CH-1 strain of Riemerella anatipestifer <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Construction of a gene-scarless deletion strain and the CH-1 strain of Riemerella anatipestifer <h2 style=";text-align:left;direction:ltr"> B739_RS01935-B739_RS07625 Construction of double gene scarless deletion strains; The Riemerella anatipestifer CH-1 strain <h2 style=";text-align:left;direction:ltr"> B739_RS01935 The construction of scarless gene deletion strains includes: (1) <h2 style=";text-align:left;direction:ltr"> B739_RS01935Upstream gene fragment <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up, <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Downstream gene fragment <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Down's amplification; (2) pBAD24:: <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up- <h2 style=";text-align:left;direction:ltr"> Cfx - <h2 style=";text-align:left;direction:ltr"> SacB Construction of recombinant plasmids; (3) pBAD24:: <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up <h2 style=";text-align:left;direction:ltr"> -Cfx - <h2 style=";text-align:left;direction:ltr"> SacB-B739_RS01935 Construction of Down recombinant plasmid; (4) <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up- <h2 style=";text-align:left;direction:ltr"> Cfx - <h2 style=";text-align:left;direction:ltr"> SacB - <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Amplification of Down fragment: using pBAD24:: B739_ <h2 style=";text-align:left;direction:ltr"> RS01935 Up <h2 style=";text-align:left;direction:ltr"> -Cfx - <h2 style=";text-align:left;direction:ltr"> SacB-B739_RS01935 Down recombination plasmid was used as template; (5) <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up- <h2 style=";text-align:left;direction:ltr"> Cfx - <h2 style=";text-align:left;direction:ltr"> SacB - <h2 style=";text-align:left;direction:ltr"> B739_RS01935 The Down fragment was naturally transformed into RA CH-1 and the positive transformants were identified to obtain the strain RA CH-1Δ <h2 style=";text-align:left;direction:ltr"> B739_RS01935 ::Up <h2 style=";text-align:left;direction:ltr"> -Cfx-SacB- Down; (6) <h2 style=";text-align:left;direction:ltr"> B739_RS01935 UP-DOWN fragment construction; (7) <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Natural transformation of UP-DOWN fragment and identification of positive transformants: strain RA CH-1Δ <h2 style=";text-align:left;direction:ltr"> B739_RS01935 ::Up <h2 style=";text-align:left;direction:ltr"> -Cfx-SacB- After Down cultivation, add <h2 style=";text-align:left;direction:ltr"> B739_RS01935 The UP-DOWN fragment was cultured and identified by PCR to obtain the strain RA CH-1Δ <h2 style=";text-align:left;direction:ltr"> B739_RS01935 .

[0007] Preferably, in step (1), specific primers containing enzyme cutting sites are designed <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up P1 (incl. <h2 style=";text-align:left;direction:ltr"> EcoR Ⅰ enzyme cutting site), <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up P2 (incl. <h2 style=";text-align:left;direction:ltr"> Kpn Ⅰ enzyme cutting site) <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up gene fragments were amplified and specific primers were designed <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Down P1 (incl. <h2 style=";text-align:left;direction:ltr"> Pst Ⅰ enzyme cutting site), <h2 style=";text-align:left;direction:ltr"> B739_RS01935 DownP2 (incl. <h2 style=";text-align:left;direction:ltr"> Hind III enzyme cutting site) <h2 style=";text-align:left;direction:ltr"> B739_RS01935Down gene fragments were amplified; The specific primer containing the enzyme cutting site <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up P1, <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up P2, B739_ <h2 style=";text-align:left;direction:ltr"> RS01935 Down P1, <h2 style=";text-align:left;direction:ltr"> B739_RS01935 The sequences of Down P2 are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively; In step (2), the <h2 style=";text-align:left;direction:ltr"> EcoR Ⅰ enzyme and <h2 style=";text-align:left;direction:ltr"> Kpn Ⅰ enzymes respectively <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up DNA fragment and pBAD24:: <h2 style=";text-align:left;direction:ltr"> Cfx - <h2 style=";text-align:left;direction:ltr"> SacB The plasmid was double-digested; The pBAD24:: <h2 style=";text-align:left;direction:ltr"> Cfx - <h2 style=";text-align:left;direction:ltr"> SacB The plasmid sequence is shown in SEQ ID NO.5; In the step (3), the <h2 style=";text-align:left;direction:ltr"> Pst Ⅰ enzyme and <h2 style=";text-align:left;direction:ltr"> Hind III enzymes respectively <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Down DNA fragment and pBAD24:: <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up- <h2 style=";text-align:left;direction:ltr"> Cfx - <h2 style=";text-align:left;direction:ltr"> SacB The recombinant plasmid was double-digested.

[0008] Preferably, in step (4), <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up- <h2 style=";text-align:left;direction:ltr"> Cfx - <h2 style=";text-align:left;direction:ltr"> SacB - <h2 style=";text-align:left;direction:ltr"> B739_RS01935 The sequence of the Down fragment is shown in SEQ ID NO.6; In step (6), design specific fusion primer fragments <h2 style=";text-align:left;direction:ltr"> B739_RS01935 UP P1, <h2 style=";text-align:left;direction:ltr"> B739_RS01935 UPP2 was amplified <h2 style=";text-align:left;direction:ltr"> B739_RS01935 UP DNA fragment, design specific fusion primer fragment <h2 style=";text-align:left;direction:ltr"> B739_RS01935 DOWN P1, B739_ <h2 style=";text-align:left;direction:ltr"> RS01935 DOWN P2 amplification <h2 style=";text-align:left;direction:ltr"> B739_RS01935 DOWNDNA fragments; then the amplified <h2 style=";text-align:left;direction:ltr"> B739_RS01935 UPDNA fragments and <h2 style=";text-align:left;direction:ltr"> B739_RS01935 DOWN DNA fragments were obtained by overlapping PCR <h2 style=";text-align:left;direction:ltr"> B739_RS01935 UP-DOWN segment; <h2 style=";text-align:left;direction:ltr"> B739_RS01935 UP P1, <h2 style=";text-align:left;direction:ltr"> B739_RS01935 UPP2, <h2 style=";text-align:left;direction:ltr"> B739_RS01935 DOWN P1, B739_<h2 style=";text-align:left;direction:ltr"> RS01935 The sequences of DOWN P2 are shown as SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0009] Preferably, in step (5), two pairs of identification primers are used in the identification of positive transformants, and the sequences of the two pairs of identification primers are shown as SEQ ID NO.1 and SEQ ID NO.8; as shown as SEQ ID NO.4 and SEQ ID NO.7; In step (7), three pairs of identification primers are used to identify positive transformants. The sequences of the three pairs of identification primers are shown in SEQ ID NO.9 and SEQ ID NO.12; shown in SEQ ID NO.7 and SEQ ID NO.13; and shown in SEQ ID NO.8 and 14.

[0010] Among them, the sequences are as follows: <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up P1 (incl. <h2 style=";text-align:left;direction:ltr"> EcoR Ⅰ restriction site): SEQ ID NO.1: CG <h2 style=";text-align:left;direction:ltr"> GAATTC GATATTAAATCACTACAAAC.

[0011] <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up P2 (incl. <h2 style=";text-align:left;direction:ltr"> Kpn Ⅰ enzyme cutting site): SEQ ID NO.2: CGG <h2 style=";text-align:left;direction:ltr"> GGTACC AAAATAGATTTAAAGTG.

[0012] <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Down P1 (incl. <h2 style=";text-align:left;direction:ltr"> Pst Ⅰ enzyme cutting site): SEQ ID NO.3:AA <h2 style=";text-align:left;direction:ltr"> CTGCAG TATAAAGCCACACTAGAGGG.

[0013] <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Down P2 (incl. <h2 style=";text-align:left;direction:ltr"> Hind III enzyme cutting site): SEQ ID NO.4: CCC <h2 style=";text-align:left;direction:ltr"> AAGCTT GTTCATAATTAAAGGTGC.

[0014] pBAD24:: <h2 style=";text-align:left;direction:ltr"> Cfx - <h2 style=";text-align:left;direction:ltr"> SacB Plasmid sequence: SEQ ID NO.5:

[0015] <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Up- <h2 style=";text-align:left;direction:ltr"> Cfx - <h2 style=";text-align:left;direction:ltr"> SacB - <h2 style=";text-align:left;direction:ltr"> B739_RS01935 Down fragment sequence: SEQ ID NO.6:

[0016] Identification primers <h2 style=";text-align:left;direction:ltr"> Cfx P1: SEQ ID NO. 7: TTTCATGTTCCATAAATCAGC.

[0017] Identification primers <h2 style=";text-align:left;direction:ltr"> SacB P2: SEQ ID NO. 8: ACGCGTCGACGTCGGTTATTTGTTAACTGTTAATTGTCCT.

[0018] <h2 style=";text-align:left;direction:ltr"> B739_RS01935 UP P1: SEQ ID NO.9: GACCCTGTAACGCTTTCTAAAGACTATACTTTAAGAGAAG.

[0019] <h2 style=";text-align:left;direction:ltr"> B739_RS01935 UP P2: SEQ ID NO. 10: CCCTCTAGTGTGGCTTTATACATAAAATAGATTTAAAGTG.

[0020] <h2 style=";text-align:left;direction:ltr"> B739_RS01935 DOWN P1: SEQ ID NO. 11: CACTTTAAATCTATTTTATGTATAAAGCCACACTAGAGGG.

[0021] <h2 style=";text-align:left;direction:ltr"> B739_RS01935 DOWN P2: SEQ ID NO. 12: AGGTAGCTAAAGCGGTAGAAATATTACATAAGAAATATCC.

[0022] Identification primers <h2 style=";text-align:left;direction:ltr"> Cfx P2: SEQ ID NO. 13: TACACCTGTTTTTGCATTCTTTT.

[0023] Identification primers <h2 style=";text-align:left;direction:ltr"> SacB P1: SEQ ID NO. 14: CTAGTCTAGACTAGTTTTTTTTTAACATTTGATTTTGTAT.

[0024] Preferably, the Riemerella anatipestifer CH-1 strain <h2 style=";text-align:left;direction:ltr"> B739_RS01935-B739_RS07625 The construction of double gene scarless deletion strains includes: (1) B739_RS07625 Upstream gene fragment B739_RS07625 Up, B739_RS07625Downstream gene fragment B739_RS07625 Down's amplification; (2) pBAD24:: B739_RS07625 Up- Cfx - SacB Construction of recombinant plasmids; (3) pBAD24:: B739_RS07625 Up -Cfx - SacB - B739_RS07625 Construction of Down recombinant plasmid; (4) B739_RS07625 Up- Cfx - SacB - B739_RS07625 Amplification of Down fragment: using pBAD24:: B739_ RS07625 Up -Cfx - SacB - B739_RS07625 Down recombination plasmid was used as template; (5) B739_RS07625 Up- Cfx - SacB - B739_RS07625 Natural transformation of Down fragment and identification of positive transformants to obtain strain RA CH-1Δ B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down; (6) B739_RS07625 UP-DOWN fragment construction and fusion; (7) B739_RS07625 Natural transformation of UP-DOWN fusion fragment and identification of positive transformants: RA CH-1Δ B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down after cultivation, add B739_RS07625 The UP-DOWN fusion fragment was cultured and identified by PCR to obtain the strain RA CH-1Δ B739_RS01935 Δ B739_RS07625 (i.e. Riemerella anatipestifer CH-1Δ B739_RS01935 Δ B739_RS07625 ).

[0025] Preferably, in step (1), specific primers containing enzyme cutting sites are designed B739_RS07625 Up P1 (incl. EcoR I restriction enzyme site), B739_RS07625 Up P2 (incl. Kpn I restriction enzyme site) B739_RS07625Up gene fragments were amplified and specific primers containing enzyme cutting sites were designed. B739_RS07625 Down P1 (incl. Sal I restriction enzyme site), B739_ RS0�625 Down P2 (incl. [[ID=۴۶]]Pst I restriction enzyme site) B739_RS07625 Down gene fragments were amplified; Specific primers containing restriction enzyme cutting sites B739_RS07625 Up P1, B739_RS07625 Up P2, B739_ RS07625 Down P1, ​ The sequences of Down P2 are shown in SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18, respectively; In step (2), the ​ I enzyme and ​ I enzymes respectively ​ Up DNA fragment and pBAD24:: ​ - ​ The plasmid was double-digested; In step (3), the ​ I enzyme and ​ I enzymes respectively ​ Down fragment and pBAD24:: ​ Up- ​ - ​ The recombinant plasmid was double-digested.

[0026] Preferably, in step (4), ​ Up- ​ - ​ - ​ The sequence of the Down fragment is shown in SEQ ID NO. 19; In step (6), design specific fusion primers ​ UP P1, ​ UP P2 were amplified ​ UPDNA fragment, design specific fusion primers ​ DownP1, B739_ ​ DownP2 was amplified ​ DOWNDNA fragments; then the amplified ​ UPDNA fragments and ​ DOWN DNA fragments were obtained by overlapping PCR ​ UP-DOWN segment; Specific fusion primers ​UP P1, ​ UP P2, ​ DownP1, ​ The sequences of DownP2 are shown in SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, and SEQ ID NO.23.

[0027] Preferably, in step (5), two pairs of identification primers are used to identify positive transformants, and the sequences of the two pairs of identification primers are shown as SEQ ID NO.15 and SEQ ID NO.8; as shown as SEQ ID NO.18 and SEQ ID NO.7; In step (7), three pairs of identification primers are used to identify positive transformants. The sequences of the three pairs of identification primers are shown in SEQ ID NO.7 and SEQ ID NO.13; shown in SEQ ID NO.8 and SEQ ID NO.14; and shown in SEQ ID NO.20 and SEQ ID NO.23.

[0028] Among them, the sequences are as follows: ​ Up P1 (incl. ​ I restriction enzyme cutting site): SEQ ID NO.15: CG ​ CAATAATTTTGATATTAGGG.

[0029] ​ Up P2 (incl. ​ I restriction enzyme cutting site): SEQ ID NO.16: GG ​ AGAATTAAAAATAGAACCGC.

[0030] ​ Down P1 (incl. ​ I restriction enzyme cutting site): SEQ ID NO. 17: ACGC ​ CATTAAAGACTAAATCTGAT.

[0031] ​ Down P2 (incl. ​ I restriction enzyme cutting site): SEQ ID NO. 18: AA ​ GGCAGATTTGGAAGCACAAC.

[0032] ​ Up- ​ - ​ - ​ Down segment: SEQ ID NO.19:

[0033] ​ UP P1: SEQ ID NO. 20: ATTTTAGAAGGTATAGAAAAGTCTTCTTTAAAAATAGGAG.

[0034] ​ UP P2: SEQ ID NO. 21: CAATCAGATTTAGTCTTTAAATAGAATTAAAAATAGAACC.

[0035] ​ DOWN P1: SEQ ID NO. 22: GGTTCTATTTTTAATTCTATTTAAAGACTAAATCTGATTG.

[0036] ​ DOWN P2: SEQ ID NO. 23: GAGATTGTGTATTTCGTTAATCAAACAAGTAATAATAG.

[0037] The third object of the present invention is to provide the application of the above-mentioned attenuated vaccine candidate strain of Riemerella anatipestifer with double gene deletion, specifically its application in preparing attenuated vaccine for Riemerella anatipestifer disease or preparing drugs for preventing and treating Riemerella anatipestifer infection.

[0038] The fourth object of the present invention is to provide an attenuated vaccine for Riemerella anatipestifer disease, comprising the attenuated vaccine candidate strain of Riemerella anatipestifer double gene deletion as described above.

[0039] Compared with the prior art, the present invention has the following advantages and beneficial effects: The attenuated strain RA CH-1Δ constructed by the present invention ​ Δ ​ After immunization of ducklings, there is no significant effect on the weight growth of ducklings, and it has good safety. ​ Δ ​ After immunization, the protection rate of ducklings against the wild-type virulent strain of RACH-1 reached 83.3%, and the immune protection effect was good. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the examples. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be considered as limiting the scope. A person of ordinary skill in the art can also derive other relevant drawings based on these drawings without inventive effort. In the drawings: ​ : ​ Up, ​ PCR amplification results of Down homologous fragments: M: Marker; Lane 1: ​ Up amplified fragment electrophoresis results; Lane 2: ​ Down amplified fragment electrophoresis results.

[0041] ​ :pBAD24:: ​ Up- ​ - ​ Construction of recombinant plasmid: M: Marker; Lane 1: ​ Up fragment identification electrophoresis results.

[0042] ​ :pBAD24:: B739_RS01935 Up- Cfx - SacB - B739_RS01935 Construction of Down recombinant plasmid: M: Marker; Lane 1: B739_RS01935 Down fragment identification electrophoresis results.

[0043] Figure 4 : B739_RS01935 Up- Cfx - SacB - B739_RS01935 Amplification of Down fragment: M: Marker; Lane 1: B739_RS01935 Up- Cfx - SacB - B739_RS01935 Down fragment amplification electrophoresis results.

[0044] Figure 5 :RA CH-1Δ B739_RS01935 ::Up- Cfx - SacB -Down PCR identification: M: Marker; Lane 1: B739_RS01935 Up- Cfx - SacB Fragment amplification electrophoresis results; Lane 2: Cfx - SacB - B739_RS01935 Down fragment amplification electrophoresis results.

[0045] Figure 6 : B739_RS01935 UP-DOWN fragment construction: M: Marker; Lane 1: B739_RS01935 UP fragment amplification electrophoresis results; Lane 2: B739_RS01935 DOWN fragment amplification electrophoresis results; Lane 3: B739_RS01935 Electrophoresis results of UP-DOWN fusion fragment.

[0046] Figure 7 :RA CH-1 Δ B739_RS01935 PCR identification: M: Marker; Lane 1: RA CH-1Δ B739_ RS01935 For template Cfx Gene identification electrophoresis results; Lane 2: RA CH-1Δ B739_RS01935 For template SacB Gene identification electrophoresis results; Lane 3: RA CH-1Δ B739_RS01935 For template B739_RS01935 Identification electrophoresis results; Lane 4: RACH-1 as template B739_RS01935 Identify the electrophoresis results.

[0047] Figure 8 : B739_RS07625 Up, B739_RS07625 PCR amplification results of Down homologous fragments: M: Marker; Lane 1: B739_RS07625 Up amplified fragment electrophoresis results; Lane 2: B739_RS07625 Down amplified fragment electrophoresis results.

[0048] Figure 9 :pBAD24 ::B739_RS07625 Up- Cfx - SacB Construction of recombinant plasmid: M: Marker; Lane 1: B739_RS07625 Up fragment identification electrophoresis results.

[0049] Figure 10 :pBAD24 ::B739_RS07625 Up- Cfx - SacB - B739_RS07625 Construction of Down recombinant plasmid: M: Marker; Lane 1: B739_RS07625 Down fragment identification electrophoresis results.

[0050] Figure 11 : B739_RS07625 Up- Cfx - SacB - B739_RS07625 Amplification of Down fragment: M: Marker; Lane 1: B739_RS07625 Up- Cfx - SacB - B739_RS07625 Down fragment amplification electrophoresis results.

[0051] Figure 12 :RA CH-1Δ B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down PCR identification: M: Marker; Lane 1: B739_RS07625 Up- Cfx - SacB Fragment amplification electrophoresis results; Lane 2: Cfx - SacB - B739_ RS07625 Down fragment amplification electrophoresis results.

[0052] Figure 13 : B739_RS07625 UP-DOWN fragment construction: M: Marker; Lane 1: B739_RS07625 UP fragment amplification electrophoresis results; Lane 2: B739_RS07625 DOWN fragment amplification electrophoresis results; Lane 3: B739_RS07625 Electrophoresis results of UP-DOWN fusion fragment.

[0053] Figure 14 :RA CH-1 Δ B739_RS01935 Δ B739_RS07625 PCR identification: M: Marker; Lane 1: RACH-1Δ B739_RS01935 Δ B739_RS07625 For template Cfx Gene identification electrophoresis results; Lane 2: RA CH-1 Δ B739_RS01935 Δ B739_RS07625 For template SacB Gene identification electrophoresis results; Lane 3: RA CH-1 Δ B739_ RS01935 Δ B739_RS07625 For template B739_RS07625 Gene identification electrophoresis results; Lane 4: RA CH-1 Δ B739_ RS01935 Δ B739_RS07625 For template B739_RS07625 Gene identification electrophoresis results.

[0054] Figure 15 : Experimental group (immune RA CH-1 B739_RS01935-B739_RS07625 Line graph of body weight on day 15 for the group (gene-deleted attenuated strain) and the blank group. DETAILED DESCRIPTION

[0055] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with examples and drawings. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0056] The present invention adopts the following method to construct a candidate strain of attenuated vaccine of Riemerella anatipestifer with double gene deletion: 1. Construction of a scarless deletion strain of Riemerella anatipestifer CH-1 B739_RS01935 1. B739_RS01935 Upstream gene fragment ( B739_RS01935 Up), B739_RS01935 Downstream gene fragment ( B739_RS01935 Down) amplification: Using the RA CH-1 genome as a reference, specific primers were designed. B739_ RS01935 Up P1 (incl. EcoR Ⅰ restriction site), B739_RS01935 Up P2 (incl. Kpn Ⅰ restriction site), B739_ RS01935 Down P1 (incl. Pst Ⅰ restriction site), B739_RS01935 Down P2 (incl. Hind III restriction site), the primer sequences are shown in SEQ ID NO.1~4. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. and amplified using RA CH-1 genome as template. B739_RS01935 Up DNA fragments and B739_RS01935 DownDNA fragments. After confirming the length of the PCR amplification product by agarose gel electrophoresis, the relevant DNA fragments were recovered using a DNA purification kit. Subsequently, the DNA fragment concentration was determined and the fragments were stored at -20°C.

[0057] 2. pBAD24:: B739_RS01935 Up- Cfx - SacB Construction of recombinant plasmid: First, use EcoR Ⅰ enzyme and Kpn Ⅰ enzyme to 1 μg B739_RS01935 Up DNA fragment and 1 μg pBAD24- Cfx - SacBThe plasmid was double-digested. Secondly, the enzyme digestion product was recovered using a DNA purification and recovery kit, and its concentration was determined. Then the two were ligated. The system required plasmid: fragment = 1:3 (substance ratio), and the connection was carried out at 16°C for 16 hours. After the connection was completed, a heat shock transformation experiment was performed. Using the transformed colony as a template, colony PCR was performed to identify the recombinant plasmid. The identification primer sequence is shown in SEQ ID NO.1~2, pBAD24- Cfx - SacB The plasmid sequence is shown in SEQ ID NO.5.

[0058] 3. pBAD24:: B739_RS01935 Up -Cfx - SacB-B739_RS01935 Construction of Down recombinant plasmid: First, use Pst Ⅰ enzyme and Hind III enzymes were respectively used to treat 1 μg B739_RS01935 Down DNA fragment and 1 μg pBAD24:: B739_ RS01935 Up- Cfx - SacB The recombinant plasmid was double-digested and the concentration was determined. After ligation, heat shock transformation experiment and PCR identification, the recombinant plasmid pBAD24:: B739_RS01935 Up -Cfx - SacB-B739_RS01935 The identification primer sequences are shown in SEQ ID NOs. 3-4.

[0059] 4. B739_RS01935 Up- Cfx - SacB - B739_RS01935 Amplification of Down fragment: using pBAD24:: B739_ RS01935 Up -Cfx - SacB - B739_RS01935 Down recombinant plasmid was used as template for PCR amplification, and the amplification primer sequences were SEQ ID NO.1 and SEQ ID NO.4. DNA purification and recovery kit was used for recovery operation to obtain B739_ RS01935 Up- Cfx - SacB - B739_RS01935 Down fragment, the fragment sequence is shown in SEQ ID NO.6.

[0060] 5. B739_RS01935 Up- Cfx - SacB - B739_RS01935 Down fragment was naturally transformed into RA CH-1 and identification of positive transformants (first homologous recombination): RA CH-1 parent strain was cultured in liquid medium until the logarithmic growth phase and the bacterial solution was adjusted to 1 OD / mL. Then, 300 μL of bacterial solution was taken and 2 μg of purified B739_RS01935 Up- Cfx - SacB - B739_RS01935 Down fragments, mix well, incubate at 37℃ for 1 hour, take 100 μL of bacterial solution, spread on 5% sterile defibrinated sheep blood plate containing 1 μg / mL cefoxitin (Cfx), and incubate at 37℃ for 24 hours. After PCR identification of the single colony, the strain RA CH-1Δ was obtained. B739_RS01935 ::Up -Cfx-SacB- Down, the sequences of the two pairs of identification primers are shown in SEQ ID NO.1 and SEQ ID NO.8; SEQ ID NO.4 and SEQ ID NO.7.

[0061] 6. B739_RS01935 UP-DOWN fragment construction: Using the RA CH-1 genome as a reference, specific fusion primer fragments were designed. B739_RS01935 UP P1 / P2 and DOWN P1 / P2 were amplified using the RA CH-1 genome as a template. B739_ RS01935 UP DNA fragments and B739_RS01935 DOWN DNA fragments. B739_RS01935 UP DNA fragments and B739_RS01935 DOWN DNA fragments were obtained by overlapping PCR B739_RS01935 UP-DOWN fragment, primer sequences are shown in SEQ ID NOs. 9 to 12.

[0062] 7. B739_RS01935 Natural transformation of UP-DOWN fusion fragment and identification of positive transformants (second homologous recombination): RA CH-1Δ B739_RS01935 ::Up- Cfx - SacB -Down were cultured in liquid medium until the logarithmic growth phase and the bacterial solution was adjusted to 1 OD / mL. Subsequently, 300 μL of bacterial solution was taken and 2 μg of purified B739_RS01935 The UP-DOWN fragment was incubated at 37°C for 1 hour, and 100 μL of the bacterial solution was spread on a 5% sterile defibrinated sheep blood plate containing 15% sucrose and incubated at 37°C for 24 hours. Single colonies that grew well on the sucrose plate were picked and subcultured on a plate without blood antibodies and a plate containing 1 μg / mL cefoxitin for 24 hours. Colonies that grew on the plate without blood antibodies and could not grow on the plate containing 1 μg / mL cefoxitin were selected as candidate strains for PCR identification, and strain RA CH-1Δ was obtained. B739_RS01935 The sequences of the three pairs of identification primers are shown in SEQ ID NO.9 and SEQ ID NO.12; SEQ ID NO.7 and SEQ ID NO.13; SEQ ID NO.8 and 14.

[0063] 2. Riemerella anatipestifer CH-1 strain B739_RS01935-B739_RS07625 Construction of double gene scarless deletion strain 1. B739_RS07625 Upstream gene fragment ( B739_RS07625 Up), B739_RS07625 Downstream gene fragment ( B739_RS07625 Down) amplification: Using the RA CH-1 genome as a reference, specific primers were designed. B739_ RS07625 Up P1 (incl. EcoR I restriction enzyme site), B739_RS07625 Up P2 (incl. Kpn I restriction enzyme site), B739_ RS07625 Down P1 (incl. Sal I restriction enzyme site), B739_RS07625 Down P2 (incl. Pst I restriction enzyme cutting site), the primer sequences are shown in SEQ ID NO.15~18. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. and amplified using RA CH-1 genome as template. B739_RS07625 Up DNA fragments and B739_RS07625 DownDNA fragments. Determine the length of the PCR amplification product by agarose gel electrophoresis and recover the relevant DNA fragments using a DNA purification kit. Subsequently, determine the DNA fragment concentration and store the fragments at -20°C.

[0064] 2. pBAD24:: B739_RS07625 Up- Cfx - SacB Construction of recombinant plasmid: First, use EcoR I enzyme and Kpn I enzyme to 1 μg B739_RS07625 Up DNA fragment and 1 μg pBAD24- Cfx - SacB The plasmid was double-digested. Secondly, the enzyme digestion product was recovered using a DNA purification and recovery kit, and its concentration was determined. Then the two were ligated. The system required plasmid: fragment = 1:3 (substance molar ratio), and the connection was carried out at 16°C for 16 hours. After the connection was completed, a heat shock transformation experiment was performed. Using the transformed colony as a template, colony PCR was performed to identify the recombinant plasmid. The identification primer sequence is shown in SEQ ID NO.15~16, pBAD24- Cfx - SacB The plasmid sequence is shown in SEQ ID NO.5.

[0065] 3. pBAD24:: B739_RS07625 Up -Cfx - SacB - B739_RS07625 Construction of Down recombinant plasmid: First, use Sal I enzyme and Pst I enzyme to 1 μg B739_RS07625 Down fragment and 1 μg pBAD24 ::B739_ RS07625 Up- Cfx - SacB The recombinant plasmid was double-digested and the concentration was determined. After ligation, heat shock transformation experiment and PCR identification, the recombinant plasmid pBAD24:: B739_RS07625 Up -Cfx - SacB-B739_RS07625 The identification primer sequences are shown in SEQ ID NOs. 17-18.

[0066] 4. B739_RS07625 Up- Cfx - SacB - B739_RS07625 Amplification of Down fragment: using pBAD24:: B739_ RS07625 Up- Cfx - SacB - B739_RS07625 Down recombinant plasmid was used as template for PCR amplification, and the amplification primer sequences were SEQ ID NO.17 and SEQ ID NO.20. DNA purification and recovery kit was used for recovery operation to obtain B739_ RS07625 Up- Cfx - SacB - B739_RS07625 Down fragment, the fragment sequence is shown in SEQ ID NO.19.

[0067] 5. B739_RS07625 Up- Cfx - SacB - B739_RS07625 Natural transformation of Down and identification of positive transformants (first homologous recombination): RA CH-1Δ B739_RS01935 The scarless deletion strain was cultured in liquid medium until the logarithmic growth phase and the bacterial suspension was adjusted to 1 OD / mL. Subsequently, 300 μL of bacterial suspension was taken and 2 μg of purified B739_RS07625 Up- Cfx - SacB - B739_RS07625 Down fragments, mix well, incubate at 37℃ for 1 hour, take 100μL of bacterial solution, spread on 5% sterile defibrinated sheep blood plate containing 1μg / mL cefoxitin, and incubate at 37℃ for 24 hours. After PCR identification of the single colony, the strain RA CH-1Δ was obtained. B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down, the sequences of the two pairs of identification primers are shown in SEQ ID NO.15 and SEQ ID NO.8; SEQ ID NO.18 and SEQ ID NO.7.

[0068] 6. B739_RS07625 UP-DOWN fragment construction and fusion: Using the RA CH-1 genome as a reference, specific fusion primers were designed. B739_RS07625 UP P1 / P2 and DOWN P1 / P2 were amplified using the RA CH-1 genome as a template. B739_RS07625 UPDNA fragments and B739_RS07625 DOWN DNA fragments. B739_RS07625 UPDNA fragments and B739_RS07625 DOWN DNA fragments were obtained by overlapping PCR B739_RS07625 UP-DOWN fragment, primer sequences are shown in SEQ ID NOs. 20 to 23.

[0069] 7. B739_RS07625 Natural transformation of UP-DOWN fusion fragment and identification of positive transformants (second homologous recombination): RA CH-1Δ B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down were cultured in liquid medium until the logarithmic growth phase and the bacterial solution was adjusted to 1 OD / mL. Subsequently, 300 μL of bacterial solution was taken and 2 μg of purified B739_ RS07625 The UP-DOWN fusion fragment was incubated at 37°C for 1 hour. 100 μL of the bacterial solution was spread on a 5% sterile defibrinated sheep blood plate containing 15% sucrose and incubated at 37°C for 24 hours. Single colonies that grew well on the sucrose plate were selected and subcultured on a plate without blood antibodies and a plate containing 1 μg / mL cefoxitin for 24 hours. Colonies that grew on the plate without blood antibodies and could not grow on the plate containing 1 μg / mL cefoxitin were selected as candidate strains for PCR identification, and the strain RA CH-1 Δ was obtained. B739_ RS01935 Δ B739_RS07625 The sequences of the three pairs of identification primers are shown in SEQ ID NO.7 and SEQ ID NO.13; SEQ ID NO.8 and 14; SEQ ID NO.20 and SEQ ID NO.23.

[0070] The present invention is described in detail below through specific examples.

[0071] 1. RA CH-1 B739_RS01935 - B739_RS07625 Method for constructing gene deletion strains 1.1 RA CH-1 B739_RS01935 Method for constructing gene deletion strains 1.1.1 、 B739_RS01935 Up, B739_RS01935 PCR amplification of Down homologous fragments Using the RA CH-1 genome as a reference, two pairs of specific primers were designed: B739_RS01935 Up P1 / P2 (i.e. B739_RS01935 Up P1 and B739_RS01935 Up P2) and Down P1 / P2 (i.e. B739_RS01935 Down P1 and B739_RS01935 Down P2). Using the RA CH-1 genome as a template, a PCR amplification reaction was performed using the following system and procedure: B739_RS01935 PCR amplification system for Up fragments

[0072] B739_RS01935 PCR amplification system for Down fragments

[0073] The reaction procedure is as follows:

[0074] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0075] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in Figure 1 The DNA fragments were recovered and their concentrations were determined using NanoDrop and stored at -20°C.

[0076] B739_RS01935 Up P1 (incl. EcoR Ⅰ enzyme cutting site): CG GAATTC GATATTAAATCACTACAAAC.

[0077] B739_RS01935 Up P2 (incl. Kpn Ⅰ enzyme cutting site): CGG GGTACC AAAATAGATTTAAAGTG.

[0078] B739_RS01935 Down P1 (incl. Pst Ⅰ enzyme cutting site): AA CTGCAG TATAAAGCCACACTAGAGGG.

[0079] B739_RS01935 Down P2 (incl. Hind Ⅲ enzyme cutting site): CCC AAGCTT GTTCATAATTAAAGGTGC.

[0080] 1.1.2, pBAD24:: B739_RS01935 Up- Cfx - SacB Construction of recombinant plasmids Pick B739_RS01935 Up and pBAD24- Cfx - SacB The plasmid was subjected to double enzyme digestion operation, and the system was as follows:

[0081] After 1 hour of reaction, the digestion product was recovered and its concentration was determined using NanoDrop. Subsequently, a ligation reaction was performed. The ligation system required n (plasmid): n (fragment) = 1:3 (substance ratio), as follows:

[0082] Incubate in a 16°C metal bath for 16 hours to perform the ligation reaction. After the ligation is completed, the transformation operation is performed. The transformation components and their purposes are as follows: (1) Competent cell group: 200 μL competent E. coli DH5α (check whether the competent cells are contaminated); (2) Double-cut group: 15 μL plasmid digestion recovery product (not treated with ligase) + 200 μL competent E. coli DH5α (to observe whether the plasmid is completely cut by the endonuclease); (3) Self-ligation group: 7.5 μL plasmid digestion recovery product + 7.5 μL Solution I ligase + 200 μL competent E. coli DH5α (observe whether the plasmid after digestion occurs self-ligation); (4) Experimental group: 15 μL ligation product + 200 μL competent E. coli DH5α (observe whether the ligation is successful).

[0083] After ice-bathing each component for 10 minutes, immediately place it in a 37°C water bath for 5 minutes. Subsequently, add 1 mL of non-resistant LB liquid medium to each component and incubate at 37°C for 1 hour. After incubation, take 200 μL of each component bacterial solution and spread it on an LB resistance plate (containing 100 μg / mL Amp). Centrifuge the remaining bacterial solution of the experimental group at 5500 rpm for 5 minutes, discard 600 μL of the supernatant, resuspend the remaining bacterial solution, and take 200 μL of it and spread it on an LB resistance plate (containing 100 μg / mL Amp). Incubate at 37°C for 12 hours. The grown single colony was inoculated onto a new LB resistance plate (containing 100 μg / mL Amp) for subculture and then performed colony PCR identification. The reaction system is as follows:

[0084] The reaction procedure is as follows:

[0085] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0086] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in Figure 2 Save the colonies that show positive bands.

[0087] B739_RS01935 Up P1 (incl. EcoR Ⅰ restriction site):CG GAATTC GATATTAAATCACTACAAAC.

[0088] B739_RS01935 Up P2 (incl. Kpn Ⅰ enzyme cutting site): CGG GGTACC AAAATAGATTTAAAGTG.

[0089] 1.1.3, pBAD24:: B739_RS01935 Up - Cfx - SacB - B739_RS01935Construction of Down recombinant plasmid Pick B739_RS01935 Down and pBAD24:: B739_RS01935 Up - Cfx - SacB The recombinant plasmid was subjected to double enzyme digestion operation, and the system was as follows:

[0090] After 1 hour of reaction, the digestion product was recovered and its concentration was determined using NanoDrop. Subsequently, a ligation reaction was performed. The ligation system required n (plasmid): n (fragment) = 1:3 (substance ratio), as follows:

[0091] Place in a 16°C metal bath for 16 hours to perform the ligation reaction. After ligation is complete, perform the transformation operation using the same method as in 1.1.2. The PCR reaction system is as follows:

[0092] The reaction procedure is as follows:

[0093] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0094] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in Figure 3 Save the colonies that show positive bands.

[0095] B739_RS01935 Down P1 (incl. Pst Ⅰ enzyme cutting site): AA CTGCAG TATAAAGCCACACTAGAGGG.

[0096] B739_RS01935 Down P2 (incl. Hind Ⅲ enzyme cutting site): CCC AAGCTT GTTCATAATTAAAGGTGC.

[0097] 1.1.4、 B739_RS01935 Up- Cfx - SacB - B739_RS01935 Amplification of Down fragment Use plasmid extraction kit to extract pBAD24:: B739_RS01935 Up - Cfx - SacB - B739_ RS01935Down recombinant plasmid. Use this plasmid as a template and dilute it to 10-20 ng / μL for PCR amplification. The reaction system is as follows:

[0098] The reaction procedure is as follows:

[0099] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0100] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in Figure 4 . Recovered and assayed using NanoDrop B739_RS01935 Up- Cfx - SacB - B739_ RS01935 The concentration of Down fragment was determined and stored at -20°C.

[0101] B739_RS01935 Up P1 (incl. EcoR Ⅰ restriction site):CG GAATTC GATATTAAATCACTACAAAC.

[0102] B739_RS01935 Down P2 (incl. Hind Ⅲ enzyme cutting site): CCC AAGCTT GTTCATAATTAAAGGTGC.

[0103] 1.1.5、 B739_RS01935 Up- Cfx - SacB - B739_RS01935 Natural transformation of Down fragment and identification of positive transformants (first homologous recombination) RA CH-1 was inoculated into TSB medium and cultured at 37°C and 180 rpm until the logarithmic growth phase (OD 600 =1.0-1.5). After adjusting the bacterial solution to 1 OD / mL, 300 μL of bacterial solution was taken and 2 μg B739_RS01935 Up- Cfx - SacB - B739_RS01935Down fragments were mixed and incubated at 37°C for 1 hour. After incubation, 100 μL of the bacterial solution was spread on a 5% sterile defibrinated sheep blood plate containing 1 μg / mL cefoxitin and incubated at 37°C for 24 hours. Single off-white colonies were selected and subcultured on new 5% sterile defibrinated sheep blood plates containing 1 μg / mL cefoxitin. The resulting colonies were identified by PCR using the following system and procedure: System (1) (Up- ​ )

[0104] System (2) ( ​ -Down)

[0105] The PCR procedure is as follows:

[0106] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0107] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in ​ The positive colonies were streaked onto new 5% sterile defibrinated sheep blood plates containing 1 μg / mL cefoxitin (Cfx), incubated at 37°C for 24 h, and the bacterial lawn was scraped with an inoculating loop into 1 mL of sterile defibrinated sheep blood and stored at -80°C to obtain RA CH-1Δ. ​ ::Up- ​ - ​ -Down.

[0108] ​ Up P1 (incl. ​ Ⅰ enzyme cutting site): CG ​ GATATTAAATCACTACAAAC.

[0109] ​ P2: ACGCGTCGACGTCGGTTATTTGTTAACTGTTAATTGTCCT.

[0110] ​ P1:TTTCATTGTTCCATAAATCAGC.

[0111] ​ Down P2 (incl. ​ Ⅲ enzyme cutting site): CCC ​ GTTCATAATTAAAGGTGC.

[0112] 1.1.6、 ​ UP-DOWN fragment construction Using the RA CH-1 genome as a reference, specific primer fragments were designed. ​ UP P1 / P2 and DOWN P1 / P2 were amplified using the RA CH-1 genome as a template. ​ UP fragment and B739_ ​ DOWN fragment. Overlap PCR was performed on the two to obtain ​ UP-DOWN fragment, primer sequences are shown in SEQ ID NOs. 9 to 12.

[0113] (1) ​ UP fragment PCR amplification system

[0114] (2) ​ PCR amplification system for DOWN fragments

[0115] Reaction procedure

[0116] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0117] (3) ​ UP fragment and ​ PCR fusion system of DOWN fragment

[0118] The reaction procedure is as follows

[0119] Note: 5 cycles of "denaturation-annealing-extension" are required.

[0120] After the reaction was completed, the reaction solution was added ​ UP P1 and ​ DOWN 2.5 μL of P2. Continue the reaction as follows:

[0121] Note: 25 cycles of "denaturation-annealing-extension" are required.

[0122] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in ​ . Recovered and assayed using NanoDrop ​The concentration of Up-Down fragments was determined and stored at -20°C.

[0123] ​ UP P1:GACCCTGTAACGCTTTCTAAAGACTATACTTTAAGAGAAG.

[0124] ​ UP P2:CCCTCTAGTGTGGCTTTATACATAAAATAGATTTAAAGTG.

[0125] ​ DOWN P1: CACTTTAAATCTATTTTATGTATAAAGCCACACTAGAGGG.

[0126] ​ DOWN P2: AGGTAGCTAAAGCGGTAGAAATATTACATAAGAAATATCC.

[0127] 1.1.7, ​ Natural transformation of the UP-DOWN fusion fragment and identification of positive transformants (second homologous recombination) RA CH-1Δ ​ ::Up- ​ - ​ -Down was inoculated into TSB medium and cultured at 37°C, 180 rpm until the logarithmic growth phase (OD 600 =1.0-1.5). After adjusting the bacterial solution to 1 OD / mL, 300 μL of bacterial solution was taken and 2 μg ​ UP-DOWN fragments were mixed and incubated at 37°C for 1 hour. After incubation, 100 μL of the bacterial solution was spread on a 5% sterile defibrinated sheep blood plate containing 15% sucrose and incubated at 37°C for 24 hours. Single colonies that grew well on the sucrose plate were selected and subcultured on a plate containing no anti-blood antibodies and a plate containing 1 μg / mL cefoxitin for 24 hours. Colonies that grew on the plate containing no anti-blood antibodies but did not grow on the plate containing 1 μg / mL cefoxitin were selected as candidate strains for PCR identification. The system and procedure are as follows: System (1)— ​ :

[0128] System (2)— ​ :

[0129] The reaction procedure is as follows:

[0130] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0131] System (3)—Target gene

[0132] The reaction procedure is as follows:

[0133] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0134] System (4)—Positive Control RA CH-1

[0135] The reaction procedure is as follows:

[0136] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0137] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in ​ The positive colonies were streaked onto new 5% sterile defibrinated sheep blood plates and incubated at 37°C for 24 h. The bacterial lawn was scraped with an inoculating loop into 1 mL of sterile defibrinated sheep blood and stored at -80°C to obtain RA CH-1Δ. B739_ ​ .

[0138] ​ P1:TTTCATTGTTCCATAAATCAGC.

[0139] ​ P2:TACACCTGTTTTTGCATTCTTTT.

[0140] ​ P1: CTAGTCTAGACTAGTTTTTTTTTAACATTTGATTTTGTAT.

[0141] ​ P2: ACGCGTCGACGTCGGTTATTTGTTAACTGTTAATTGTCCT.

[0142] ​ UP P1:GACCCTGTAACGCTTTCTAAAGACTATACTTTAAGAGAAG.

[0143] ​ DOWN P2: AGGTAGCTAAAGCGGTAGAAATATTACATAAGAAATATCC.

[0144] 1.2 RA CH-1 B739_RS01935 - B739_RS07625 Method for constructing gene deletion strains 1.2.1、 B739_RS07625 Up, B739_RS07625 PCR amplification of Down homologous fragments Using the RA CH-1 genome as a reference, two pairs of specific primers were designed: B739_RS07625 Up P1 / P2 and Down P1 / P2. Using the RA CH-1 genome as a template, PCR amplification was performed using the following system and procedure: (1) B739_RS07625 PCR amplification system of Up homologous fragments

[0145] (2) B739_RS07625 PCR amplification system for Down fragments

[0146] The reaction procedure is as follows:

[0147] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0148] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in Figure 8 The DNA fragments were recovered and their concentrations were determined using NanoDrop and stored at -20°C.

[0149] B739_RS07625 Up P1 (incl. EcoR I restriction enzyme site): CG GAATTC CAATAATTTTGATATTAGGG.

[0150] B739_RS07625 Up P2 (incl. Kpn I restriction enzyme site): GG GGTACC AGAATTAAAAATAGAACCGC.

[0151] B739_RS07625 Down P1 (incl. Sal I restriction enzyme site): ACGC GTCGACCATTAAAGACTAAATCTGAT.

[0152] B739_RS07625 Down P2 (incl. Pst I restriction enzyme site): AA CTGCAG GGCAGATTTGGAAGCACAAC.

[0153] 1.2.2, pBAD24:: B739_RS07625 Up- Cfx - SacB Construction of recombinant plasmids Pick B739_RS07625 Up and pBAD24- Cfx - SacB The plasmid was subjected to double enzyme digestion operation, and the system was as follows:

[0154] After 1 hour of reaction, the digestion product was recovered and its concentration was determined using NanoDrop. Subsequently, a ligation reaction was performed. The ligation system required n (plasmid): n (fragment) = 1:3 (substance ratio), as follows:

[0155] Incubate in a 16°C metal bath for 16 hours to perform the ligation reaction. After the ligation is completed, the transformation operation is performed. The transformation components and their purposes are as follows: (1) Competent cell group: 200 μL competent E. coli DH5α (check whether the competent cells are contaminated); (2) Double-cut group: 15 μL plasmid digestion recovery product (not treated with ligase) + 200 μL competent E. coli DH5α (to observe whether the plasmid is completely cut by the endonuclease); (3) Self-ligation group: 7.5 μL plasmid digestion recovery product + 7.5 μL Solution I ligase + 200 μL competent E. coli DH5α (observe whether the plasmid after digestion occurs self-ligation); (4) Experimental group: 15 μL ligation product + 200 μL competent E. coli DH5α (observe whether the ligation is successful).

[0156] After ice-bathing each component for 10 minutes, immediately place it in a 37°C water bath for 5 minutes. Subsequently, add 1 mL of non-resistant LB liquid medium to each component and incubate at 37°C for 1 hour. After incubation, take 200 μL of each component bacterial solution and spread it on an LB resistance plate (containing 100 μg / mL Amp). Centrifuge the remaining bacterial solution of the experimental group at 5500 rpm for 5 minutes, discard 600 μL of the supernatant, resuspend the remaining bacterial solution, and take 200 μL of it and spread it on an LB resistance plate (containing 100 μg / mL Amp). Incubate at 37°C for 12 hours. The grown single colony was inoculated onto a new LB resistance plate (containing 100 μg / mL Amp) for subculture and then performed colony PCR identification. The reaction system is as follows:

[0157] The reaction procedure is as follows:

[0158] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0159] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in Figure 9 Save the colonies that show positive bands.

[0160] B�39_RS07625 Up P1 (incl. EcoR I restriction enzyme site): CG GAATTC CAATAATTTTGATATTAGGG.

[0161] B739_RS07625 Up P2 (incl. Kpn I restriction enzyme site): GG GGTACC AGAATTAAAAATAGAACCGC.

[0162] 1.2.3、pBAD24:: B739_RS07625 Up - Cfx - SacB - B739_RS07625 Construction of Down recombinant plasmid Pick B739_RS07625 Down and pBAD24:: B739_RS07625 Up - Cfx - SacB The recombinant plasmid was subjected to double enzyme digestion operation, and the system was as follows:

[0163] After 1 hour of reaction, the digestion product was recovered and its concentration was determined using NanoDrop. Subsequently, a ligation reaction was performed. The ligation system required n (plasmid): n (fragment) = 1:3 (substance ratio), as follows:

[0164] Place in a 16°C metal bath for 16 hours to perform the ligation reaction. After the ligation is completed, perform the transformation operation using the same method as in 1.2.2. The reaction system is as follows:

[0165] The reaction procedure is as follows:

[0166] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0167] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in Figure 10 Save the colonies that show positive bands.

[0168] B739_RS07625 Down P1 (incl. Sal I restriction enzyme site): ACGC GTCGAC CATTAAAGACTAAATCTGAT.

[0169] B739_RS07625 Down P2 (incl. Pst I restriction enzyme site): AA CTGCAG GGCAGATTTGGAAGCACAAC.

[0170] 1.2.4、 B739_RS07625 Up- Cfx - SacB - B739_RS07625 Amplification of Down homologous fragments Use plasmid extraction kit to extract pBAD24:: B739_RS07625 Up - Cfx - - B739_ SacB Down recombinant plasmid. Use this plasmid as a template and dilute it to 10-20 ng / μL for PCR amplification. The reaction system is as follows:

[0171] The reaction procedure is as follows:

[0172] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0173] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in RS07625 . Recovered and assayed using NanoDrop Figure 11 Up- B739_RS07625 - Cfx - B739_ SacB The concentration of Down fragment was determined and stored at -20°C.

[0174] RS07625 Up P1 (incl. B739_RS07625 I restriction enzyme site): CG [[ID=H59]]EcoR CAATAATTTTGATATTAGGG.

[0175] GAATTC Down P2 (incl. B739_RS07625 I restriction enzyme site): AA Pst GGCAGATTTGGAAGCACAAC.

[0176] 1.2.5, CTGCAG Up- B739_RS07625 - Cfx - SacB Natural transformation of Down fragment and identification of positive transformants (first homologous recombination) RA CH-1Δ B739_RS07625 Inoculate into TSB medium and culture at 37°C, 180 rpm until the logarithmic growth phase (OD 600 =1.0-1.5). After adjusting the bacterial solution to 1 OD / mL, 300 μL of bacterial solution was taken and 2 μg B739_RS01935 Up- B739_RS07625 - Cfx - SacB Down fragments were mixed and incubated at 37°C for 1 hour. After incubation, 100 μL of the bacterial solution was spread on a 5% sterile defibrinated sheep blood plate containing 1 μg / mL cefoxitin and incubated at 37°C for 24 hours. Single off-white colonies were selected and subcultured on new 5% sterile defibrinated sheep blood plates containing 1 μg / mL cefoxitin. The resulting colonies were identified by PCR using the following system and procedure: System (1) (Up- B739_RS07625 )

[0177] System (2) ( SacB -Down)

[0178] PCR procedure

[0179] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0180] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in Cfx The positive colonies were streaked onto new 5% sterile defibrinated sheep blood plates containing 1 μg / mL cefoxitin (Cfx), incubated at 37°C for 24 h, and the bacterial lawn was scraped with an inoculating loop into 1 mL of sterile defibrinated sheep blood and stored at -80°C to obtain RA CH-1Δ. Figure 12 Δ B739_RS01935 ::Up- B739_RS07625 - Cfx -Down.

[0181] SacB Up P1 (incl. I restriction enzyme site): CG B739_RS07625 CAATAATTTTGATATTAGGG.

[0182] EcoR P2: ACGCGTCGACGTCGGTTATTTGTTAACTGTTAATTGTCCT.

[0183] GAATTC P1:TTTCATTGTTCCATAAATCAGC.

[0184] SacB Down P2 (incl. Cfx I restriction enzyme site): AA B739_RS076 GGCAGATTTGGAAGCACAAC.

[0185] 1.2.6, Pst UP-DOWN fragment construction and fusion Using the RA CH-1 genome as a reference, specific primer fragments were designed. CTGCAG UP P1 / P2 and DOWN P1 / P2 were amplified using the RA CH-1 genome as a template. B739_RS07625 UP fragment and B739_ B739_RS07625 DOWN fragment. Overlap PCR was performed on the two to obtain B739_RS07625 UP-DOWN fragment, primer sequences are shown in SEQ ID NOs. 20 to 23.

[0186] (1) RS07625 UP fragment PCR amplification system

[0187] (2) B739_RS07625 PCR amplification system for DOWN fragments

[0188] The reaction procedure is as follows:

[0189] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0190] (3) B739_RS07625 UP fragment and B739_RS07625 PCR fusion system of DOWN fragment

[0191] The reaction procedure is as follows:

[0192] Note: 5 cycles of "denaturation-annealing-extension" are required.

[0193] After the reaction was completed, the reaction solution was added B739_RS07625 UP P1 and B739_RS07625 DOWN 2.5 μL of P2. Continue the reaction as follows:

[0194] Note: 25 cycles of "denaturation-annealing-extension" are required.

[0195] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in B739_RS07625 . Recovered and assayed using NanoDrop B739_RS07625 The concentration of Up-Down fragments was determined and stored at -20°C.

[0196] UP P1:ATTTTAGAAGGTATAGAAAAGTCTTCTTTAAAAATAGGAG.

[0197] Figure 13 UP P2:CAATCAGATTTAGTCTTTAAATAGAATTAAAAATAGAACC.

[0198] B739_RS07625 DOWN P1: GGTTCTATTTTTAATTCTATTTAAAGACTAAATCTGATTG.

[0199] B739_RS07625 DOWN P2: GAGATTGTGTATTTCGTTAATCAAACAAGTAATAATAG.

[0200] 1.2.7, B739_RS07625 Natural transformation of the UP-DOWN fusion fragment and identification of positive transformants (second homologous recombination) Δ [[ID=10^3]]B739_RS07625 Δ B739_RS07625 ::Up- B739_RS07625 - B739_RS07625 -Down was inoculated into TSB medium and cultured at 37°C and 180 rpm until the logarithmic growth phase (OD 600 =1.0-1.5). After adjusting the bacterial solution to 1 OD / mL, 300 μL of bacterial solution was taken and 2 μg [[ID=10^7]]B739_RS07625 Up-Down fragments were mixed and incubated at 37°C for 1 hour. After incubation, 100 μL of the bacterial solution was spread on a 5% sterile defibrinated sheep blood plate containing 15% sucrose and incubated at 37°C for 24 hours. Single colonies that grew well on the sucrose plate were subcultured on a plate containing no anti-blood antibodies and a plate containing 1 μg / mL cefoxitin for 24 hours. Colonies that grew on the plate containing no anti-blood antibodies and did not grow on the plate containing 1 μg / mL cefoxitin were selected as candidate strains for PCR identification. The system and procedure are as follows: System (1)— Cfx :

[0201] System (2)— SacB :

[0202] The reaction procedure is as follows:

[0203] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0204] System (3)—Target gene

[0205] The reaction procedure is as follows:

[0206] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0207] System (4)——Positive Control RA CH-1

[0208] The reaction procedure is as follows:

[0209] Note: The "denaturation-annealing-extension" cycle needs to be cycled 30 times in the entire PCR process.

[0210] After the PCR reaction is completed, take 5 μL of the reaction solution, add 1 μL of DNA Loading Buffer and perform spotting operation. The results of agarose gel electrophoresis are shown in B739_RS07625 The positive colonies were streaked onto new 5% sterile defibrinated sheep blood plates and incubated at 37°C for 24 h. The bacterial lawn was scraped with an inoculating loop into 1 mL of sterile defibrinated sheep blood and stored at -80°C to obtain RA CH-1Δ. B739_ Cfx Δ SacB .

[0211] Figure 14 P1:TTTCATTGTTCCATAAATCAGC.

[0212] RS01935 P2:TACACCTGTTTTTGCATTCTTTT.

[0213] P1: CTAGTCTAGACTAGTTTTTTTTTAACATTTGATTTTGTAT.

[0214] B739_RS07625 P2: ACGCGTCGACGTCGGTTATTTGTTAACTGTTAATTGTCCT.

[0215] Cfx UP P1:ATTTTAGAAGGTATAGAAAAGTCTTCTTTAAAAATAGGAG.

[0216] Cfx DOWN P2: GAGATTGTGTATTTCGTTAATCAAACAAGTAATAATAG.

[0217] 2. RA CH-1 SacB SacB B739_RS07625 B739_RS07625 B739_RS01935 - B739_RS07625 It should be noted that there seems to be an error in the "B�39_RS07625" in line 26, which is likely a typo. It is translated as "B739_RS07625" according to the context. Also, there is an unclear "B739_RS076 " in line 85, which is tentatively translated as "B739_RS07625" for the sake of translation integrity. And "10^3" and "10^7" in lines 103 and 107 are likely incorrect notations and are translated as normal numbers "103" and "107" respectively. Safety evaluation of a double-gene deletion strain as a candidate gene deletion vaccine (effects on weight gain in ducklings) The 3-day-old ducklings were divided into 2 groups: Group 1, 12 ducklings were immunized with RA CH-1Δ B739_RS01935 Δ B739_ RS07625The second group of 12 ducklings did not receive any treatment. They were fed with sufficient antibiotic-free feed and drinking water. The weight of each duck was measured daily for 14 consecutive days. The results showed that compared with the control group, the weight of the ducks treated with RA CH-1Δ B739_ RS01935 Δ B739_RS07625 Double gene deletion strains immunized ducklings will not significantly affect their normal growth ( Figure 15 ).

[0218] 3. RA CH-1 B739_RS01935-B739_RS07625 Evaluation of the immune protection effect of double gene deletion strains as candidate gene deletion vaccines The 3-day-old ducklings were divided into 2 groups: Group 1 was treated with 1×10 9 CFU RA CH-1Δ B739_RS01935 Δ B739_ RS07625 Immunize 3-day-old ducklings and challenge with 100×LD 50 The wild strain of RA CH-1; Group 2, 3-day-old ducklings without any treatment, and 7 days later challenged with 100×LD 50 The RA CH-1 wild strain (LD50 of the RA CH-1 wild strain was determined to be 2.24×10 8 The morbidity and mortality of ducks within 10 days after challenge were calculated to evaluate the protection rate of the attenuated candidate vaccine. B739_RS01935 Δ B739_RS07625 The protection rate against wild-type RA CH-1 after immunization was 83.3% ( Table 2 ).

[0219] Table 1. RA CH-1 B739_RS01935-B739_RS07625 Determination of the immune protection of ducklings by double gene deletion strain

[0220] The above results show that this attenuated strain is safe and reliable in application, has a high protection effect against toxins, and can be used as a candidate strain for the development of new gene-deficient vaccines.

[0221] The above specific implementation methods further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A candidate strain of a double-gene-deleted attenuated vaccine against Riemerella anatipestifer, characterized in that: Riemerella anatipestifer CH-1 strain B739_RS01935-B739_RS07625 The double-gene deleted attenuated vaccine candidate strain, the preservation number is CCTCC NO: M20251080, the preservation date is May 15, 2025, and it is preserved in the China Center for Type Culture Collection. The preservation unit address is Wuhan University, Wuhan, China.

2. The method for constructing a candidate strain of a double-gene-deleted attenuated vaccine of Riemerella anatipestifer according to claim 1, characterized in that: Including Riemerella anatipestifer CH-1 strain B739_RS01935 Construction of a gene-scarless deletion strain and the CH-1 strain of Riemerella anatipestifer B739_RS01935-B739_RS07625 Construction of double gene scarless deletion strains; The Riemerella anatipestifer CH-1 strain B739_RS01935 The construction of scarless gene deletion strains includes: (1) B739_RS01935 Upstream gene fragment B739_RS01935 Up, B739_RS01935 Downstream gene fragment B739_ RS01935 Down's amplification; (2) pBAD24:: B739_RS01935 Up- Cfx - SacB Construction of recombinant plasmids; (3) pBAD24:: B739_RS01935 Up -Cfx - SacB-B739_RS01935 Construction of Down recombinant plasmid; (4) B739_RS01935 Up- Cfx - SacB - B739_RS01935 Amplification of Down fragment: using pBAD24:: B739_ RS01935 Up -Cfx - SacB-B739_RS01935 Down recombination plasmid was used as template; (5) B739_RS01935 Up- Cfx - SacB - B739_RS01935 The Down fragment was naturally transformed into RA CH-1 and the positive transformants were identified to obtain the strain RA CH-1Δ B739_RS01935 ::Up -Cfx-SacB- Down; (6) B739_RS01935 UP-DOWN fragment construction; (7) B739_RS01935 Natural transformation of UP-DOWN fragment and identification of positive transformants: strain RA CH-1Δ B739_RS01935 ::Up -Cfx-SacB- After Down cultivation, add B739_RS01935 The UP-DOWN fragment was cultured and identified by PCR to obtain the strain RA CH-1Δ B739_RS01935 .

3. The method for constructing a candidate strain of a double-gene-deleted attenuated vaccine of Riemerella anatipestifer according to claim 2, characterized in that: In step (1), specific primers containing enzyme cutting sites were designed B739_RS01935 Up P1, B739_ RS01935 Up P2 pair B739_RS01935 Up gene fragments were amplified and specific primers were designed B739_ RS01935 Down P1, B739_RS01935 Down P2 pair B739_RS01935 Down gene fragments were amplified; The specific primer containing the enzyme cutting site B739_RS01935 Up P1, B739_RS01935 Up P2, B739_ RS01935 Down P1, B739_RS01935 The sequences of Down P2 are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively; In step (2), the EcoR Ⅰ enzyme and Kpn Ⅰ enzymes respectively B739_RS01935 Up DNA fragment and pBAD24:: Cfx - SacB The plasmid was double-digested; The pBAD24:: Cfx - SacB The plasmid sequence is shown in SEQ ID NO.5; In the step (3), the Pst Ⅰ enzyme and Hind III enzymes respectively B739_RS01935 Down DNA fragment and pBAD24:: B739_RS01935 Up- Cfx - SacB The recombinant plasmid was double-digested.

4. The method for constructing a candidate strain of a Riemerella anatipestifer double-gene deleted attenuated vaccine according to claim 2, characterized in that: In step (4), B739_RS01935 Up- Cfx - SacB - B739_RS01935 The sequence of the Down fragment is shown in SEQ ID NO.6; In step (6), design specific fusion primer fragments B739_RS01935 UP P1, B739_RS01935 UPP2 was amplified B739_RS01935 UP DNA fragment, design specific fusion primer fragment B739_RS01935 DOWN P1, B739_ RS01935 DOWN P2 amplification B739_RS01935 DOWNDNA fragments; then the amplified B739_RS01935 UPDNA fragments and B739_RS01935 DOWN DNA fragments were obtained by overlapping PCR B739_RS01935 UP-DOWN segment; B739_RS01935 UP P1, B739_RS01935 UPP2, B739_RS01935 DOWN P1, B739_RS01935 The sequences of DOWNP2 are shown as SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, respectively.

5. The method for constructing a candidate strain of a double-gene-deleted attenuated vaccine of Riemerella anatipestifer according to claim 2, characterized in that: In step (5), two pairs of identification primers are used to identify positive transformants. The sequences of the two pairs of identification primers are shown in SEQ ID NO.1 and SEQ ID NO.8; and in SEQ ID NO.4 and SEQ ID NO.7; In step (7), three pairs of identification primers are used to identify positive transformants. The sequences of the three pairs of identification primers are shown in SEQ ID NO.9 and SEQ ID NO.12; shown in SEQ ID NO.7 and SEQ ID NO.13; and shown in SEQ ID NO.8 and 14.

6. The method for constructing a candidate strain of a double-gene-deleted attenuated vaccine of Riemerella anatipestifer according to claim 2, characterized in that: The Riemerella anatipestifer CH-1 strain B739_RS01935-B739_RS07625 The construction of double gene scarless deletion strains includes: (1) B739_RS07625 Upstream gene fragment B739_RS07625 Up, B739_RS07625 Downstream gene fragment B739_ RS07625 Down's amplification; (2) pBAD24:: B739_RS07625 Up- Cfx - SacB Construction of recombinant plasmids; (3) pBAD24:: B739_RS07625 Up -Cfx - SacB - B739_RS07625 Construction of Down recombinant plasmid; (4) B739_RS07625 Up- Cfx - SacB - B739_RS07625 Amplification of Down fragment: using pBAD24 ::B739_ RS07625 Up -Cfx - SacB - B739_RS07625 Down recombination plasmid was used as template; (5) B739_RS07625 Up- Cfx - SacB - B739_RS07625 Natural transformation of Down fragment and identification of positive transformants to obtain strain RA CH-1Δ B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down; (6) B739_RS07625 UP-DOWN fragment construction and fusion; (7) B739_RS07625 Natural transformation of UP-DOWN fusion fragment and identification of positive transformants: RA CH-1Δ B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down after cultivation, add B739_RS07625 The UP-DOWN fusion fragment was cultured and identified by PCR to obtain the strain RA CH-1 Δ B739_RS01935 Δ B739_RS07625.

7. The method for constructing a candidate strain of a double-gene-deleted attenuated vaccine of Riemerella anatipestifer according to claim 6, characterized in that: In step (1), specific primers containing enzyme cutting sites were designed B739_RS07625 Up P1, B739_ RS07625 Up P2 pair B739_RS07625 Up gene fragments were amplified and specific primers containing enzyme cutting sites were designed. B739_RS07625 Down P1, B739_RS07625 Down P2 pair B739_RS07625 Down gene fragments were amplified; Specific primers containing restriction enzyme cutting sites B739_RS07625 Up P1, B739_RS07625 Up P2, B739_ RS07625 Down P1, B739_RS07625 The sequences of Down P2 are shown in SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18, respectively; In step (2), the EcoR I enzyme and Kpn I enzymes respectively B739_RS07625 Up DNA fragment and pBAD24:: Cfx - SacB The plasmid was double-digested; In step (3), the Sal I enzyme and Pst I enzymes respectively B739_RS07625 Down fragment and pBAD24:: B739_ RS07625 Up- Cfx - SacB The recombinant plasmid was double-digested.

8. The method for constructing a candidate strain of a double-gene-deleted attenuated vaccine of Riemerella anatipestifer according to claim 6, characterized in that: In step (4), B739_RS07625 Up- Cfx - SacB - B739_RS07625 The sequence of the Down fragment is shown in SEQ ID NO. 19; In step (6), design specific fusion primers B739_RS07625 UP P1, B739_RS07625 UP P2 were amplified separately B739_RS07625 UPDNA fragment, design specific fusion primers B739_RS07625 DownP1, B739_RS07625 DownP2 was amplified B739_RS07625 DOWNDNA fragments; then the amplified B739_RS07625 UPDNA fragments and B739_RS07625 DOWN DNA fragments were obtained by overlapping PCR B739_RS07625 UP-DOWN segment; Specific fusion primers B739_RS07625 UP P1, B739_RS07625 UP P2, B739_RS07625 DownP1, B739_RS07625 The sequences of DownP2 are shown in SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, and SEQ ID NO.

23.

9. The method for constructing a candidate strain of a double-gene-deleted attenuated vaccine of Riemerella anatipestifer according to claim 6, characterized in that: In step (5), two pairs of identification primers are used to identify positive transformants. The sequences of the two pairs of identification primers are shown in SEQ ID NO. 15 and SEQ ID NO. 8; and in SEQ ID NO. 18 and SEQ ID NO. 7; In step (7), three pairs of identification primers are used to identify positive transformants. The sequences of the three pairs of identification primers are shown in SEQ ID NO.7 and SEQ ID NO.13; shown in SEQ ID NO.8 and SEQ ID NO.14; and shown in SEQ ID NO.20 and SEQ ID NO.

23.

10. Use of the attenuated vaccine candidate strain of Riemerella anatipestifer with double gene deletion according to claim 1 in preparing an attenuated vaccine for Riemerella anatipestifer disease or in preparing a drug for preventing and treating Riemerella anatipestifer infection.

11. A attenuated vaccine for Riemerella anatipestifer disease, characterized in that: The invention comprises the candidate strain of attenuated vaccine of Riemerella anatipestifer with double gene deletion as claimed in claim 1.

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